Innate immune cells lack the exquisite specificity of the adaptive immune system, yet in order to respond in a measured way they must be able to tailor their response to the specific pathogen. These cells have therefore evolved pattern recognition receptors (PRRs) that recognize conserved molecular patterns characteristic of the microbe, which are not found within the host. Recognition of the specific combination of patterns by the PRRs allows the host cell to phenotype the invader and to respond appropriately. The Toll- like receptors, or TLRs, are the prototypic pattern recognition receptors. They recognize a broad spectrum of molecules including IPS (TLR4), dsRNA (TLRS), ssRNA (TLR7 and 8), CpG DNA (TLR9) and flagellin (TLRS). TLR 2 acts as a heterodimer with TLR1 to recognize triacylated lipoprotein, and with TLR6 to recognized diacylated lipoprotein. Current dogma suggests that although these two heterodimers have a distinct repertoire of recognition, they activate identical signaling pathways through the adaptors MyD88 and TIRAP. We have a number of lines of evidence that indicates that this is not so. In this proposal we use a combination of traditional approaches together with the tools of systems biology to delineate the mechanism and consequences of differential signaling by TLR2/1 and TLR2/6. The work will be carried out in the context of three broad aims. First, we will use a variety of biochemical and genetic approaches to identify the molecular determinants that confer differential signaling between TLR2/1 and 2/6. Second, we will use a combination of computational tools, high throughput global technologies and more traditional molecular biological approaches to define the transcriptional regulatory networks that result from differential signaling by TLR2/1 and TLR2/6. Finally, we will assess the biologic consequences of differential signaling by TLR2/1 and TLR2/6 in shaping adaptive immune responses in mice. Lay summary. The proteins that are being investigated have a pivotal role in regulating the inflammatory and immune response in people. They instruct the body to effectively combat infectious disease, and an understanding of their function will permit us to make better vaccines. However, these molecules are a two-edged sword. When they function incorrectly they lead to inflammatory and autoimmune diseases. Understanding how this happens will lead to the design and production of better drugs for diseases such as rheumatoid arthritis.